Remedies for dissease with hypermyotonia

ABSTRACT

An M toxin of type A botulinum toxin (HA-negative substance) and a mixture of L toxin and LL toxin (HA-positive substance) are compared and examined in inhibitory action for neuromuscular transmission and therapeutic index. As a result, it is found that M toxin of type A botulinum toxin has characteristics of: 1) having an excellent inhibitory action for neuromuscular transmission; 2)showing a high therapeutic index; 3) showing a low antigenicity and 4) suffering from little reduction in efficacy even after repeatedly administered, compared with the mixture of L toxin and LL toxin. Owing to these characterics, the M toxin of type A botulinum toxin is particularly useful as a therapeutic agent for diseases caused by hypermyotonia such as strabismus, blepharospasm, facial spasms, spasmodic torticollis, paralysis after cerebral apoplexy, infantile cerebral paralysis, spasmodic phonopathy, headache such as migraine, chronic pain such as lumbago, stiff shoulder, muscular relaxation disorder accompanied with onset of Parkinson&#39;s disease or multiple sclerosis, myofascial pain syndrome, masticatory spasm, chronic anal fissure, urinary inconsistency, grinding of teeth, facial myokymia, tic, topical dystonia and wrinkles.

BACKGROUND ART

The present invention relates to therapeutic agents for diseases causedby hypermyotonia where an M toxin of type A botulinum toxin as an activeingredient and, more particularly, it relates to therapeutic agents forstrabismus, blepharospasm, hemifacial spasms, spasmodic torticollis,paralysis after cerebral apoplexy, infantile cerebral paralysis,spasmodic phoropathy, headache, lumbago, neck pain, back pain, stiffshoulder, muscular relaxation disorder accompanied by Parkinson'sdisease or multiple sclerosis, myofascial pain syndrome, masticatoryspasm, chronic anal fissure, urinary inconsistency, grinding teeth,facial myokymia, tic, topical dystonia, wrinkles, etc.

TECHNICAL FIELD

Clostridium botulinum is an obligate anaerobic Gram-positive bacillusand it has been known that toxin produced by Clostridium botulinum has ahigh affinity to terminal area of peripheral nerve and causes botulism,where a main symptom is flaccid paralysis of whole-body skeletal muscle.

Toxin produced by Clostridium botulinum is classified into seven, A toG, according to the difference in antigenicity and may be furtherdivided into M-, L- and LL toxins according to the difference in thestructure of non-toxic protein bonding to neurotoxin. In JP-A-2003-9897,there is a description that, when a botulinum toxin solution containingM-, L- and LL toxins is passed through a lactose column, it is able tobe separated into an M toxin (hemagglutinin-negative substance orHA-negative substance) showing no hemagglutinating activity and L- andLL toxins (hemagglutinin-positive substances or HA-positive substances)showing a hemagglutinating activity.

It has been known that, in type A botulinum toxin, there are threetypes, i.e. an M toxin (HA-negative substance) having molecular weightof about 300,000 to which non-toxic component having no hemagglutinatingactivity to neurotoxin is bonded, an L toxin (HA-positive substance)having molecular weight of about 500,000 to which non-toxic componenthaving hemagglutinating activity to an M toxin and an LL toxin(HA-positive substance) having molecular weight of about 900,000 wheretwo L toxins are associated with each other.

Incidentally, strabismus is caused by a breakdown of a balance in strainamong sphincter muscles and blepharospasm is a disease where opening ofeye becomes difficult by involuntary contraction of orbicular muscles ofthe eye. Facial spasms is a disease where facial muscle involuntarilyshrinks due to irritability accentuation of facial nerves whilespasmodic torticollis is a disease where abnormality is caused in headdue to abnormal tension of muscle of neck. Masticatory spasm is adisease where involuntary movement happens in the lower jaw and movementof pulling up the lower jaw in both sides (closing the mouth) happensinvoluntarily. Grinding of the teeth is a symptom like masticatory spasmand is a phenomenon during sleeping being thought to be a kind of sleepdisorder. Spasmodic phonopathy is a disease where involuntarycontraction happens in motor muscle of vocal cord whereby normalutterance is unable to be done. In facial myokymia, spasm of muscularfillets happens in a part of muscle bundle of the face in groups and, inthe skin or the surface of mucous membrane, swaying or swellingsustained involuntary movement is observed. Tic is sometimes calledTourette's syndrome and is a disease where spasmodic, dramatic andsudden muscle spasm happens. Its examples are nictiation, wry face, headshaking and crying. Chronic anal fissure is accompanied by over-tensionof anal sphincter caused by repetitive anal fissure and is a diseaseresulting in anal stenosis due to loss of elasticity of anus. Inimminent urinary incontinence, a part of muscles of bladder strainsexcessively in an involuntary manner whereby a strong urination feelingis apt to be noted and it is a disease to urinate regardless of one'swill. Myofascial pain syndrome is a disease where, as a result of acutedisorder or repetitive overloaded stress (too much use) of muscle, hardstiffness-like part (strain belt) is formed in muscle and strong painoccurs and it has been known that muscle strain of hand and foot isexcessively promoted after cerebral apoplexy or as a result of onset ofinfantile cerebral palsy, Parkinson's disease or multiple sclerosis. Ithas been also known that headache such as migraine occurs chronicallywhen strain of muscles of neck or shoulder is excessively promoted andthat strain of muscle is abnormally promoted by too much use of muscleor a sustained poor posture and, as a result, chronic pain or stiff necksuch as lumbago, neck pain and back pain is induced. Wrinkles aregenerated by shrinking of muscle and examples of wrinkles on the faceare wrinkles between the brow, wrinkles at corner of the eye andwrinkles at nose root. As such, all of those strabismus, blepharospasm,facial spasms, spasmodic torticollis, paralysis after cerebral apoplexy,infantile cerebral paralysis, spasmodic phonopathy, headache such asmigraine, chronic pain such as lumbago, stiff neck, muscular relaxationdisorder accompanied with onset of Parkinson's disease or multiplesclerosis, myofascial pain syndrome, masticatory spasm, chronic analfissure, urinary inconsistency, grinding of teeth, facial myokymia, tic,topical dystonia and wrinkles are caused by hypermyotonic action.

With regard to utilization of botulinum toxin for treatment of diseasescaused by hypermyotonia, its examples are that botulinum toxin is usedfor treatment of strabismus in Ophthalmology, 87, 1044-1049 (1980) andthat, in J. Fr. Ophthalmol. 13, 259-264 (1990), it is used for treatmentof blepharospasm. In JP-A-8-511536, there is disclosed a method wherebotulinum toxin is administered until a decrease in clinical response iscaused and, after that, other botulinum toxin is administered to treatthe disease of nerve muscle. In JP-A-8-511537, there is disclosed amethod where at least two of botulinum toxins of type A to type G arejointly administered whereby the disease of nerve muscle is treated. In“Therapy with Botulinum Toxin”, Marcel Dekker, New York, 1994, pages577-595, it is reported that botulinum toxin is effective for treatmentof wrinkles between brow.

Actually, as a botulinum toxin effective for the treatment ofblepharospasm, one-side face spasm, spasmodic strabismus, wrinkles,etc., BOTOX® (manufactured by Allergan) where an LL toxin of botulinumtoxin is an active ingredient has been sold in the market.

However, in any of the aforementioned literatures, there is neitherdescription nor suggestion at all for application of an M toxin of typeA botulinum toxin (HA-negative substance) for the treatment of diseasescaused by hypermyotonia such as strabismus, blepharospasm and wrinkles.

DISCLOSURE OF THE INVENTION

Although botulinum toxin has been known as a drug for mitigation ofmuscle strain, the toxin itself is a drug having strong toxicity.Therefore, as a result of its side effect, botulinum toxin may causesystemic malaise by mitigation of muscle strain. Particularly when thereis a mistake for its dose, severe side effect happens whereby it isdesirable that dose of botulinum toxin is made as little as possible. Inaddition, a problem that, when botulinum toxin is repeatedlyadministered, its efficacy is attenuated has been pointed out and thephenomenon as such is thought to be dependent upon production ofantibody to the toxin. Accordingly, a therapeutic agent which does notinduce antibody production and does not lower its effect even whenadministered repeatedly has been demanded. Although BOTOX® where an LLtoxin of botulinum toxin is an active ingredient has been sold in themarket already, there has been a demand for far better products in viewof pharmaceutical effect and side effect.

The present inventors have carried out intensive studies paying theirattention to the constituting components (M toxin, L toxin and LL toxin)of type Abotulinum toxin and, as a result, they have found that an Mtoxin (HA-negative substance) having molecular weight of about 300,000being bonded to non-toxic component showing no agglutination activity toneurotoxin has a better inhibiting activity to nerve muscle transmissionthan a mixture of an L toxin having molecular weight of about 500,000being bonded to non-toxic component showing agglutination activity to anM toxin and an LL toxin having molecular weight of about 900,000(HA-positive substance), has a therapeutic index of five times higherthan commercially available BOTOX®, rarely produces an antibody and isable to maintain its effect even upon repetitive administration wherebyan M toxin of type A botulinum toxin is particularly effective as atherapeutic agent for various diseases caused by hypermyotonia wherebythe present invention has been achieved.

The present invention relates to an inhibitor for neuromusculartransmission or a therapeutic agent for the treatment of diseases causedby hypermyotonia comprising an M toxin (HA-negative substance) of type Abotulinum toxin as an active ingredient.

The M toxin of type A botulinum toxin according to the present inventionhas an excellent inhibiting activity for neuromuscular transmission anda high therapeutic index, rarely induces the antibody production andshows little attenuation of the effect even upon repetitiveadministration whereby it is useful as a therapeutic agent for diseasescaused by hypermyotonia such as strabismus, blepharospasm, facialspasms, spasmodic torticollis, paralysis after cerebral apoplexy,infantile cerebral paralysis, spasmodic phonopathy, headache such asmigraine, chronic pain such as lumbago, stiff shoulder, muscularrelaxation disorder accompanied with onset of Parkinson's disease ormultiple sclerosis, myofascial pain syndrome, masticatory spasm, chronicanal fissure, urinary inconsistency, grinding of teeth, facial myokymia,tic, topical dystonia and wrinkles. Examples of wrinkles are those onthe face such as wrinkles between the brow caused by shrinking of facialmuscle, wrinkles at corner of the eye, wrinkles at nose root andwrinkles at the chin.

Although details thereof will be mentioned under the item ofpharmacological tests, an inhibitive activity for nerve transmission ofan M toxin (HA-negative substance) of type A botulinum toxin wasevaluated by conducting a test on muscular tension. In the musculartension test, specimens of diaphragm and phrenic nerve of mice were usedand, as a comparative control drug, a mixture of L- and LL toxins oftype A botulinum toxin was used. By conducting a test on grip of hindpaw of mice, therapeutic index (TD₂₀/ED₅₀) of an M toxin (HA-negativesubstance) of type A botulinum toxin was evaluated (here, ED₅₀ is a 50%effective dose while TD₂₀ is a 20% toxic dose). Further, antigenicity ofan M toxin of type A botulinum toxin was evaluated by a test for themeasurement of antibody value. In the grip test of hind paw andmeasurement test for antibody value as such, mice were used and, ascomparative control drugs, a mixture of L- and LL-toxins of type Abotulinum toxin and a commercially available BOTOX® were used. Althoughthose tests using comparative control drugs as such are standard inconducting in the same molar ratio, biological activity (titer) of thetoxin is used as a standard because it is difficult to specify themolecular weight of a mixture of L- and LL toxins. Thus, titer of thetoxin is able to be expressed by an i.p. LD₅₀ value (50% lethal dose byintraperitoneal administration) and the test was conducted at the dosewhere the i.p. LD₅₀ values were the same.

Although an M toxin having the same titer as that of a mixture of L- andLL toxins was used, it has been found as a result of muscular tensiontest that the M toxin achieves an inhibitory activity for nervetransmission about 10 times as high as that of a mixture of L- and LLtoxins. It is usual to predict that toxins having the same titer achievethe same effect but the aforementioned finding is entirely contrary tosuch a prediction. As a result of a grip test of hind paw, it has beenalso found that the therapeutic index (TD₂₀/ED₅₀) of an M toxin of typeA botulinum toxin is about five-fold that of the commercially availableBOTOX®. As a result of the test for measurement of antibody value, ithas been further found that antigenicity of an M toxin of type Abotulinum toxin is lower than that of BOTOX® and that an M toxin of typeA botulinum toxin has less reduction in the effect even upon repeatedadministration as compared with BOTOX®. From those results, it is likelythat, when an M toxin of type A botulinum toxin is used, far highertherapeutic effect is achieved and occurrence of side effects such assystemic malaise is able to be effectively suppressed.

With regard to an M toxin of type A botulinum toxin according to thepresent invention, when incubation is carried out using a strain whichdoes not produce an HA-positive toxin such as 7I03-H, 7I05-H, Chiba-H,Kyoto-F and 804-1H among the type A Clostridium botulinum, it ispossible to produce only M toxin of type A botulinum toxin whereby anoperation for the separation of L- and LL toxins can be omitted. It isalso possible that, when a botulinum toxin solution containing M-, L-and LL toxins is passed through an ion-exchange column or a gelfiltration column, only M toxin having no agglutination activity isseparated from the mixture.

An M toxin of type A botulinum toxin does not have agglutinationactivity (HA-negative) and its molecular weight is within a range of200,000 to 400,000. On the contrary, L- and LL toxins of type Abotulinum toxin have agglutination activity (HA-positive) and theirmolecular weights are 500,000 or higher. Accordingly, an M toxin of typeA botulinum toxin is able to be clearly distinguished from L- and LLtoxin thereof.

Dose of an M toxin of type A botulinum toxin according to the presentinvention is able to be appropriately determined depending upon theobject diseases and there is no particular limitation therefor. When,however, side effect by toxin is taken into consideration, it ispreferred to be 0.01 to 500 unit(s)/site per treatment and, morepreferably, it is 0.5 to 300 unit(s)/site.

It is preferred that the therapeutic agent for diseases caused byhypermyotonia in accordance with the present invention is administeredto muscle which is an acting site of the botulinum toxin. The dosageform thereof is mostly injection preparation and is able to be made intopharmaceutical preparation using a widely used art.

The injection preparation of the present invention is able to beprepared by addition of additives such as an osmotic pressure adjustingagent (e.g., sodium chloride) and a buffer (e.g., sodium phosphate).

It is preferred that pH of the injection preparation of the presentinvention is adjusted at 4.0 to 7.5 and it is preferred that an osmoticpressure ratio is adjusted at near 1.0.

The therapeutic agent for diseases caused by hypermyotonia according tothe present invention can be injected by a commonly conductedintramuscular injection.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph which shows the relation between lethal activity andinhibitive activity for neuromuscular transmission in mice when a testsolution and a comparative solution were used.

BEST MODE FOR CARRYING OUT THE INVENTION

Production examples, pharmacological tests and preparation examples aredescribed as hereunder and those examples are intended for betterunderstanding of the present invention and do not limit the scope of thepresent invention.

1. Production of the Toxin for the Test

The toxin for the test of type A botulinum toxin was produced by amethod of Sakaguchi (Sakaguchi, G. (1983): Clostridium botulinum toxins.Pharmac. Ther., 19, 165-94) with partial modifications.

(1) PRODUCTION EXAMPLE 1 Production of Toxin of an M Toxin)

Spore cell solution of type A 7I03 strain (a strain which produces “Mtoxin”) of Clostridium botulinum stored by freezing was inoculated to apre-incubation medium (cooked meat medium) and incubated at 30° C. for 2days. The pre-incubation medium was planted to a peptone-yeastextract-glucose medium (PYG medium) and incubated at 30° C. for 3 days.

Next, 3N H₂SO₄ solution was added to the culture solution to conduct aprecipitation with an acid, the culture solution was adjusted to pH 3.5and allowed to stand at room temperature for one night. On the next day,it was centrifuged (9200×g, 20 minutes, 4° C.) and the resultingprecipitate was dissolved in 0.2M phosphate buffer (pH 6.0).

The dissolved solution was adjusted to pH 6.0 and stirred at 37° C. for1 hour to extract the toxin. The extract was centrifuged (9,200×g,20minutes, 4° C.), its supernatant liquid was collected and 0.2Mphosphate buffer (pH 6.0) containing 2% of protamine was added to give aprecipitate. This was centrifuged (9,200×g, 15 minutes, 4° C.) to give asupernatant liquid. To this supernatant liquid was added a saturatedammonium sulfate solution (390 g/L) so as to make the sulfate 60% (w/v)and the mixture was allowed to stand for one night at 4° C. to conduct asalting-out. On the next day, centrifugal separation (9,200×g, 15minutes, 4° C.) was conducted, the obtained precipitate was dissolved in0.05M acetate buffer (pH 4.2, 0.2M NaCl) and the obtained solution wasdialyzed with a permeable membrane against 0.05M acetate buffer (pH 4.2,0.2M NaCl) for one night. After completion of the dialysis, it wascentrifuged (13,700×g, 15 minutes, 4° C.) and the obtained supernatantliquid was subjected to ion-exchange using a column [SP-Sepharose FastFlow (Amersham)] equilibrated with 0.05M acetate buffer (pH 4.2, 0.2MNaCl). This was subjected to a linear gradient so as to give 0.05Macetate buffer (pH 4.2, 0.7M NaCl) finally and, as a result, impuritiesand toxin were eluted.

A toxin fraction was collected with a fraction collector and the portionwhich is to be collected as “M toxin” was determined on the basis oftoxicity measurement, OD value and electrophoretic images. The collectedfraction was concentrated by ultrafiltration (Amicon YM 30) and, inorder to raise its purity, the concentrate was passed through a gelfiltration column [Sephadex G-200 (Pharmacia)] equilibrated with 0.05Macetate buffer (pH 6.0, 0.2M NaCl) and the collected toxin fraction wasconcentrated by ultrafiltration (Amicon YM 30) to obtain an originalliquid (protein concentration: 1.78 mg/ml; biological activity: 2.0×10⁷i.p. LD₅₀/ml) of “M toxin” which is a type A botulinum toxin for apharmacological test. Incidentally, with regard to the biologicalactivity (titer), a 50% lethal dose (i.p. LD₅₀) after intraperitonealadministration to mice was used as an index.

(2) PRODUCTION EXAMPLE 2 Production of a Mixture of L Toxin and LL Toxin

The same operation as in Production Example 1 was carried out usingClostridium botulinum type A 62A strain (strain which produces “amixture of M toxin, L toxin and LL toxin”) instead of Clostridiumbotulinum A 7I03H strain to give an original liquid (proteinconcentration: 1.99 mg/ml; biological activity: 1.3×10⁷ i.p. LD₅₀/ml) of“a mixture of L toxin and LL toxin” which is a type A botulinum toxinfor a pharmacological test. A toxin fraction was collected with afraction collector and the portion which is to be collected as “amixture of L toxin and LL toxin” was determined on the basis of toxicitymeasurement, OD value and electrophoretic images.

2. Pharmacological Test

(1) Comparison of Inhibitive Activity for Neuromuscular TransmissionBased on Muscular Tension Test of Specimens of Diaphragm and PhrenicNerve of Mice

In order to compare the pharmacological activities of “M toxin” and “amixture of L toxin and LL toxin”, a muscular tension test was carriedout using diaphragm and phrenic nerve specimens of mice (strain: ddY;sex: male).

(Preparation of Test Solution)

The M toxin of Production Example 1 was diluted with 20 mM Trishydrochloride buffer containing 0.02% bovine serum albumin (pH 7.4; 150mM NaCl) to prepare a test solution (3.4×10⁵ i.p. LD₅₀/ml)

(Preparation of Comparative Solution)

A mixture of L toxin and LL toxin of Production Example 2 was dilutedwith 20 mM Tris hydrochloride buffer containing 0.02% bovine serumalbumin (pH 7.4; 150 mM NaCl) to prepare a test solution (5.5×10⁵ i.p.LD₅₀/ml).

(Measuring Method)

Pentobarbital (50 mg/kg) was intraperitoneally administered to mice toeuthanize them, the thorax was opened and right and left phrenic nerveswere ligated at the height of thymus. Right and left phrenic nerves werecarefully exfoliated from the surrounding connective tissue untildiaphragm and then cutting was also done for abdominal side whereupondiaphragm and the twelfth rib were excised in a united manner wherephrenic nerve was attached thereto. The excised diaphragm and phrenicnerve specimen was cut into one half in a water tank kept at 37° C. andeach half diaphragm piece was fixed to a tissue-supporting apparatususing a silk yarn being passed through the twelfth rib.

Next, the phrenic nerve was passed through a platinum electrode loop andtransferred to a tissue bath where the diaphragm specimen was kept at37° C. An end of silk yarn where another end thereof was ligated todiaphragm central tendon was connected to an isometric tensiontransducer and the diaphragm-phrenic nerve specimen was suspended in aKrebs solution. Rectangular wave where connecting time was 10 msec andvoltage was 1 V was applied from a platinum electrode loop to phrenicnerve with a frequency of 0.25 Hz and shrinking tension of diaphragminduced by electric stimulation to the nerve was amplified with atension amplifier and recorded with a pen recorder with a lapse of time.Load at a rest stage of about 4 g was applied to the diaphragm-phrenicnerve specimen and an observation was conducted every 15 to 20 minuteswhile exchanging the Krebs solution in the tissue bath for 1 to 2hour(s) until induced tension and base line became stable without anytreatment.

After confirming that base line and tension were stable, each of thetest solution and the comparative solution was added to the Krebssolution in the tissue bath and attenuation of the tension induced bythe toxin was recorded. After completion of the experiment, tension wasanalyzed for each of diaphragm and phrenic nerve specimens. As an indexfor attenuation of tension due to the action of the toxin, time fromaddition of the toxin until attenuation of the induced tension to anextent of 1/e of that immediately before the treatment with the toxinor, in other words, contraction tension which was recoded with elapse oftime was plotted and an approximate line was determined whereby time (τ)for treating with toxin required for attenuation to an extent of 1/e ofthe contraction tension before treatment with toxin was determined andused as an index for inhibitive activity for neuromuscular transmission(incidentally, the smaller the τ value, the higher the neuromusculartransmission inhibiting activity). From those results, FIG. 1 shows therelation between lethal action to mice and inhibitory activity ofneuromuscular transmission when the test solution and the comparativesolution were used and Table 1 shows the inhibitory activity of thecomparative solution when the inhibitory activity for neuromusculartransmission of the test solution in the same lethal dose for micewas 1. Incidentally, each plot for the test solution in FIG. 1 is a meanvalue of 4 to 5 cases and each plot for the comparative solution thereinis a mean value of 2 to 3 cases. TABLE 1 Inhibitory Activity forNeuromuscular Transmission Test Solution 1.00 Comparative Solution 0.10

(Result)

From FIG. 1 and Table 1, an inhibitory activity for neuromusculartransmission of the M toxin is about 10-fold that of a mixture of Ltoxin and LL toxin.

(2) Comparative test for Therapeutic Index Based on Grip Test of HindPaw of Mice

In order to compare the pharmaceutical effect (ED₅₀: 50% effective dose)and the toxicity (TD₂₀: 20% toxic dose) for “M toxin”, “a mixture of Ltoxin and LL toxin” and “BOTOX® (manufactured by Allergan)”, a grip testby hind paw of mice (strain: ddY; sex: male) was carried out.

(i) Study of Pharmaceutical Effect (ED₅₀)

(Preparation of Test Solutions)

The M toxin of Production Example 1 was diluted with a physiologicalsaline containing 0.1% of human serum albumin to prepare five testsolutions having different concentrations (1.2, 4, 12, 40 and 120 i.p.LD₅₀/ml).

(Preparation of Comparative Solution A)

A mixture of the L toxin and LL toxin of Production Example 2 wasdiluted with a physiological saline containing 0.1% of human serumalbumin to prepare five “comparative solution A”s having differentconcentrations (1.2, 4, 12, 40 and 120 i.p. LD_(50/)ml) in the samemanner as in the test solutions.

(Preparation of Comparative Solution B)

BOTOX® was diluted with a physiological saline containing 0.1% of humanserum albumin to prepare five “comparative solution B”s having differentconcentrations (1.2, 4, 12, 40 and 120 i.p. LD₅₀/ml) in the same manneras in the test solution.

(Measuring Method)

To gastrocnemius of right hind paw of mice was administered each ofsolvent (a physiological saline containing 0.1% of human serum albumin),test solution, comparative solution A and comparative solution B at thedose of 250 μl/kg (dose in terms of toxin for test solution, comparativesolution A and comparative solution B each was 0.3, 1, 3, 10 and 30 i.p.LD₅₀/kg). After 6 hours and 1, 2, 3, 7, 14 and 21 day(s) from theadministration, grip of right hind paw was measured using a gripstrength meter for small animals (ten animals per group). When the gripof the solvent administration group at each measuring time was defined100, the grip after the administration of each toxin was determined.Using the value at the stage where lowering in the grip in eachadministration group was maximum, ED₅₀ values of test solution,comparative solution A and comparative solution B were calculated (Table2).

(ii) Study of Toxicity (TD₂₀)

(Preparation of Test Solution)

The M toxin of Production Example 1 was diluted with a physiologicalsaline containing 0.1% of human serum albumin to prepare four testsolutions having different concentrations (20, 50, 100 and 150 i.p.LD₅₀/ml)

(Preparation of Comparative Solution A)

A mixture of the L toxin and LL toxin of Production Example 2 wasdiluted with a physiological saline containing 0.1% of human serumalbumin to prepare four test solutions having different concentrations(20, 50, 100 and 150 i.p. LD₅₀/ml) in the same manner as in the testsolutions.

(Preparation of Comparative Solution B)

BOTOX® was diluted with a physiological saline containing 0.1% of humanserum albumin to prepare four test solutions having differentconcentrations (20, 50, 100 and 150 i.p. LD₅₀/ml) in the same manner asin the test solutions.

(Measuring Method)

To quadriceps of right hind paw of mice was administered each ofsolvent, test solution, comparative solution A and comparative solutionB at the dose of 500 μl/kg (dose in terms of toxin for test solution,comparative solution A and comparative solution B each was 10, 25, 50and 75 i.p. LD₅₀/kg) After 1, 2, 3, 7 and 14 day(s) from theadministration, grip of left hind paw was measured using a grip strengthmeter (six to ten animals per group). This method measures the degree ofmuscle relaxation of the toxin in muscle of left hind paw which isanother muscle whereto the toxin was leaked from quadriceps of righthind paw which is the administration site. When the grip of the solventadministration group at each measuring time was defined 100, the gripafter the administration of each toxin was determined. Using the valueat the stage where lowering in the grip in each administration group wasmaximum, TD₂₀ values of test solution, comparative solution A andcomparative solution B were calculated (Table 2).

(iii) Therapeutic Index (TD₂₀/ED₅₀)

Ratio of the ED₅₀ value for expression of pharmaceutical effect to theTD₂₀ value for expression of toxicity for each of test solution,comparative solution A and comparative solution B was calculated and theresulting ratio was defined as a therapeutic index. The therapeuticindex is expressed as TD₂₀/ED₅₀. When the therapeutic index is higher,the deviating width between the dose showing the efficacy and the doseshowing the toxicity is bigger. As a result, it means that a drug havinghigh therapeutic index has a high usefulness as a drug. The results areshown in Table 2. TABLE 2 ED₅₀ TD₂₀ Therapeutic (i.p. LD₅₀/kg) (i.p.LD₅₀/kg) Index Test Solution 0.57 37.3 65.4 Comparative Solution A 0.8817.0 19.3 Comparative Solution B 1.27 16.2 12.8(Results)

It is apparent from Table 2 that the ED₅₀ value of the M toxin is lessthan that of a mixture of L toxin and LL toxin and that of BOTOX®whereby the M toxin has higher pharmaceutical effect than the mixture ofL toxin and LL toxin and BOTOX® and that the TD₂₀ value of the M toxinis twice as bigger than those of a mixture of L toxin and LL toxin andBOTOX® whereby the M toxin has far lower toxicity than the mixture of Ltoxin and LL toxin and BOTOX® The therapeutic index of the M toxin (testsolution) is 3.4-fold that of a mixture of L toxin and LL toxin(comparative solution A) and is 5.1-fold that of BOTOX® (comparativesolution B).

(3) Comparison of Antigenicity Based on the Measurement Test of AntibodyValue

Comparison and study were carried out to check whether the antibodyproduction was observed after repetitive administration of “M toxin”, “amixture of L toxin and LL toxin” and “BOTOX® (manufactured byAllergan)”.

(Preparation of Test Solution)

The M toxin of Production Example 1 was diluted with a physiologicalsaline containing 0.1% of human serum albumin to prepare a test solution(100 i.p. LD₅₀/ml)

(Preparation of Comparative Solution A)

A mixture of the L toxin and LL toxin of Production Example 2 wasdiluted with a physiological saline containing 0.1% of human serumalbumin to prepare a comparative solution A (100 i.p. LD₅₀/ml) in thesame manner as in the test solution.

(Preparation of Comparative Solutions B)

BOTOX® was diluted with a physiological saline containing 0.1% of humanserum albumin to prepare a comparative solution B (100 i.p. LD₅₀/ml) inthe same manner as in the test solution.

(Measuring Method)

To quadriceps of right hind paw of mice was administered each of solvent(physiological saline containing 0.1% of human serum albumin), testsolution, comparative solution A and comparative solution B at the doseof 500 μl/kg (dose in terms of toxin for test solution, comparativesolution A and comparative solution B each was 25 i.p. LD₅₀/kg) at therate of every three weeks and three times in total. After 14 days or 15days from the third administration, plasma was collected from the mice(13 to 19 animals per group) and combined with the plasma collected fromorbital vein before administration of the toxin and the obtained mixturewas subjected to a measurement of antibody value by an ELISA method.Antibody was considered to be produced in the mouse where the absorbanceratio of the plasma collected after administration of toxin to thatcollected before the administration was 10 or more and the rate of themice producing the antibody was calculated (Table 3). TABLE 3 Rate ofMice wherein Antibody was Produced (%) Test Solution 16 ComparativeSolution A 29 Comparative Solution B 31

(Result)

As shown in Table 3, the rate of mice wherein antibody was produced inthe group to which the M toxin was administered was about one half ofthe rate in the group to which a mixture of L toxin and LL toxin wasadministered or in the group to which BOTOX® was administered.Accordingly, antigenicity of the M toxin was lower than that of L toxin,LL toxin or toxin contained in BOTOX®.

(4) Comparison of Attenuating Rate of the Effect by RepetitiveAdministration

In a grip test by hind paw of mice, it was studied whether the effectwas attenuated by repetitive administration of “M toxin”, “a mixture ofL toxin and LL toxin” and “BOTOX® (manufactured by Allergan)”.

(Preparation of Test Solution)

The M toxin of Production Example 1 was diluted with a physiologicalsaline containing 0.1% of human serum albumin to prepare a test solution(2 i.p. LD₅₀/ml)

(Preparation of Comparative Solution A)

A mixture of the L toxin and the LL toxin of Production Example 2 wasdiluted with a physiological saline containing 0.1% of human serumalbumin to prepare a comparative solution A (2 i.p. LD₅₀/ml) in the samemanner as in the test solution.

(Preparation of Comparative Solutions B)

BOTOX® was diluted with a physiological saline containing 0.1% of humanserum albumin to prepare a comparative solution B (2 i.p. LD₅₀/ml) inthe same manner as in the test solution.

(Measuring Method)

Gastrocnemius of right hind paw of mice was administered each of solvent(a physiological saline containing 0.1% of human serum albumin), testsolution, comparative solution A and comparative solution B at the doseof 7.5 μl (dose in terms of toxin for test solution, comparativesolution A and comparative solution B each was 0.015 i.p. LD₅₀). After1, 2 and 3 day(s) from the administration, grip of right hind paw wasmeasured using a grip strength meter for small animals. After 28 daysfrom the first administration, the second administration was carried out(administration method and dose were the same as those in the firsttime) and grip of right hind paw was measured after 1, 2 and 3 day(s)from the administration in the same manner as in the firstadministration (11 or 12 animals per group). When the grip of thesolvent administration group at each measuring time was defined 100, thegrip after the administration of each toxin was determined. With regardto each of them at the first and the second administrations, total(1+2+3 day(s) after administration) of the three measured day for eachmouse was calculated. After that, mean value of the difference betweenthe value at the first administration and that at the secondadministration was calculated for each total value whereupon anattenuating rate for the effect was calculated (Table 4). Incidentally,when the value was bigger, the effect at the second administration wasmore attenuated. TABLE 4 Attenuating Rate (%) of Effect Test Solution  5Comparative Solution A 48 Comparative Solution B 26

(Result)

As will be apparent from Table 4, as compared with the attenuating rateof effect at the second administration of the M toxin, the attenuatingrate of effect of a mixture of L toxin and LL toxin was about 10-foldand that of BOTOX® was about 5-fold. From those results, it is apparentthat the M toxin hardly causes attenuation of the effect upon repetitiveadministration as compared with L toxin, LL toxin and BOTOX®.

3. Example of Pharmaceutical Preparation

Injection Preparation

A common example of pharmaceutical preparation of an injectionpreparation according to the present invention is as follows.Formulation 1 (in 100 mL) M toxin of type A botulinum toxin 1,000 unitsHuman serum albumin 75 mg Physiological saline q. s.

INDUSTRIAL APPLICABILITY

As apparent from the result of the pharmacological test, an M toxin oftype A botulinum toxin (HA-negative substance) has higher inhibitoryaction for neuromuscular transmission than a mixture of L toxin and LLtoxin (HA-positive substance) and also has a therapeutic index of 3- to5-fold that of a mixture of L toxin and LL toxin (HA-positive substance)or a commercially available BOTOX®. Moreover, it hardly induces antigenproduction and, even when administered repeatedly, its attenuation inthe effect is small and, accordingly, it is particularly useful as atherapeutic agent for diseases caused by hypermyotonia such asstrabismus, blepharospasm, facial spasms, spasmodic torticollis,paralysis after cerebral apoplexy, infantile cerebral paralysis,spasmodic phonopathy, headache such as migraine, chronic pain such aslumbago, stiff shoulder, muscular relaxation disorder accompanied withonset of Parkinson's disease or multiple sclerosis, myofascial painsyndrome, masticatory spasm, chronic anal fissure, urinaryinconsistency, grinding of teeth, facial myokymia, tic, topical dystoniaand wrinkles.

1-6. (canceled)
 7. A method for inhibiting neuromuscular transmissioncomprising administering to a patient an effective amount of an M toxin(HA-negative substance) of type A botulinum toxin.
 8. A method fortreating a disease caused by hypermyotonia comprising administering to apatient an effective amount of an M toxin (HA-negative substance) oftype A botulinum toxin.
 9. The method for treating according to claim 8,wherein the disease caused by hypermyotonia is strabismus,blepharospasm, hemifacial spasms, spasmodic torticollis, paralysis aftercerebral apoplexy, infantile cerebral paralysis, spasmodic phonopathy,headache, lumbago, neck pain, back pain, stiff shoulder, muscularrelaxation disorder accompanied by Parkinson's disease or multiplesclerosis, myofascial pain syndrome, masticatory spasm, chronic analfissure, urinary inconsistency, grinding of teeth, facial myokymia, tic,topical dystonia or wrinkles.
 10. The method for treating according toclaim 8, wherein molecular weight of the M toxin of type A botulinumtoxin is 200,000 to 400,000.
 11. The method for treating according toclaim 9, wherein molecular weight of the M toxin of type A botulinumtoxin is 200,000 to 400,000.
 12. The method for treating according toclaim 8, wherein the M toxin of type A botulinum toxin is produced by astrain of Clostridium botulinum type A 7I03-H, Clostridium botulinumtype A Chiba or Clostridium botulinum type A Kyoto-F.
 13. The method fortreating according to claim 9, wherein the M toxin of type A botulinumtoxin is produced by a strain of Clostridium botulinum type A 7I03-H,Clostridium botulinum type A Chiba or Clostridium botulinum type AKyoto-F.
 14. The method for treating according to claim 10, wherein theM toxin of type A botulinum toxin is produced by a strain of Clostridiumbotulinum type A 7I03-H, Clostridium botulinum type A Chiba orClostridium botulinum type A Kyoto-F.
 15. The method for treatingaccording to claim 11, wherein the M toxin of type A botulinum toxin isproduced by a strain of Clostridium botulinum type A 7I03-H, Clostridiumbotulinum type A Chiba or Clostridium botulinum type A Kyoto-F.
 16. Themethod for treating according to claim 8, wherein the M toxin isadministered in the form of an injection.
 17. The method for treatingaccording to claim 9, wherein the M toxin is administered in the form ofan injection.
 18. The method for treating according to claim 10, whereinthe M toxin is administered in the form of an injection.
 19. The methodfor treating according to claim 11, wherein the M toxin is administeredin the form of an injection.
 20. The method for treating according toclaim 12, wherein the M toxin is administered in the form of aninjection.
 21. The method for treating according to claim 13, whereinthe M toxin is administered in the form of an injection.
 22. The methodfor treating according to claim 14, wherein the M toxin is administeredin the form of an injection.
 23. The method for treating according toclaim 15, wherein the M toxin is administered in the form of aninjection.